Congenital heart disease is the most common of all birth defects (Hoffman and Kaplan, 2002). For successful prevention of or therapeutic intervention in congenital heart disease, it is of utmost importance to understand its etiology. Toward this goal, an understanding of the origin of specific cardiac lineages and their interactions with each other is critical. Understanding the origin and properties of cardiac progenitors is also important for the development of cardiac stem cell therapies for both congenital and adult heart disease.
Recent work has defined two fields of cardiac progenitors, dubbed the primary and secondary, or anterior heart fields (Kelly and Buckingham, 2002). The primary heart field is believed to give rise to the atria and ventricles of the heart, while the secondary or anterior field is believed to give rise to the outflow tract. The secondary field is believed to reside anterior and dorsal to the heart at the early linear heart tube stage. Initial evidence that the outflow tract of the heart was not present in the linear heart tube came from a series of in vivo lineage studies performed in chick embryos by de la Cruz and colleagues from 1977 onward (de la Cruz, 2000). These studies demonstrated that the outflow tract was not present at the linear heart tube stage, but did not indicate where the outflow tract came from at a later stage.
Recently, the source of the outflow tract has been addressed by studies from three different laboratories, two performed in chick embryos, and one performed in mouse embryos (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). Results of these studies demonstrated that some cells in the outflow tract originate from splanchnic mesoderm adjacent to the pharyngeal endoderm. The extent of the contribution, and the boundaries of the “secondary” or “anterior” heart field could not be definitively assessed from results of these experiments.
Stem cells have been defined in many different ways. However, the main principles include: (1) self-renewal, or the ability to generate daughter cells with characteristics similar to the initiating mother cell; (2) multi-lineage differentiation of a single cell; and (3) in vivo functional reconstitution of damaged tissue.
The Embryonic Stem (ES) cells, first obtained from mouse (Evans and Kaufmann, 1981) and more recently from non-human primates and human blastocysts (Thomson, et al., 1998), display all three characteristics. ES cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be propagated indefinitely in an undifferentiated state. Both mouse and human ES cell-lines have been maintained continuously in culture for more than 300 cell doublings. ES cells differentiate into all somatic cell types when injected into a blastocyst and form mature progeny cells of all three embryonic germ layers in vitro. Finally, all differentiated progeny of ES cells are functional cells, as mice generated by tetraploid embryo complementation are viable. Although ES cells have been isolated from humans, their use in research as well as their therapeutic potential is encumbered by ethical considerations.
Most adult stem cells also fulfil the stem cell criteria mentioned above, even though the degree of self-renewal and differentiation is less than that seen for ES cells. The best studied adult stem cell, the hematopoietic stem cell (HSC) (Weissman, 2000), undergoes in vivo self-renewing cell divisions, differentiates at the single cell level into all mature blood elements, and functionally repopulates the bone marrow of myeloablated animals and humans. Other adult stem cells have been more recently defined and are, therefore, less well studied. However, neural stem cells (NSC) (Gage, 2000), mesenchymal stem cells (MSC) (Jiang, et al., 2002) and epidermal stem cells (Toma, et al., (2001) all fulfil these basic criteria. Other cells also termed stem cells, such as angioblasts or endothelial stem cells (Rafii, et al., 1994), display all the required characteristics, except that they differentiate only into a single type of cells.
Over the last few years a plethora of literature has been published indicating that cells from a given tissue might be capable of differentiating into cells of a different tissue “Stem cell plasticity” is a new term that has been used to describe the recent observations that greater potential might persist in postnatal adult stem cells than previously expected. The majority of studies using bone marrow (BM), or peripheral blood enriched for HSC were based on in vivo transplantation of either sex-mismatched cells or genetically marked cells, and detection of donor cells was based on the presence of the Y-chromosome or the marker gene. There are excellent reviews of the pitfalls involved in the detection of donor cells using either marking system (Tisdale and Dunbar, 2002). Differentiation, not only into hematopoietic cells, but also into cells with characteristics of skeletal muscle (Gussoni, et al., 1999), cardiac muscle (Orlic, et al., 2001), endothelium (Jackson, et al., 2001), neuroectoderm (Brazelton, et al., 2000) and endodermal cells (Krause, et al., 2001), including hepatocytes, has been described.
In 80% of these studies, fresh BM cells were transplanted without prior in vitro culture, so that the question of whether the cell with plasticity can undergo self-renewal could not be assessed. In the majority of these studies, non-purified populations of cells or cells purified to partial homogeneity were transplanted, therefore making it impossible to study the clonal origin of differentiated cells or the tissue of origin of cells with characteristics of a second tissue. Finally, these studies depended on phenotypic characteristics to define differentiation into cells different from the tissue of origin, but have yet to demonstrate that the cells of the second tissue have functional characteristics of that lineage.
Thus there is need in the art for new and better methods of in vitro expansion and propagation of undifferentiated cardiac progenitor cells. The method includes culturing isolated undifferentiated progenitor cells that express Islet1 under conditions sufficient for progenitor cell growth. study of